1. Field of the Invention
The present invention relates to a new class of compounds of pyridinium series and to their use in treatment of diabetes and related illnesses. More particularly the invention relates to compounds of this series, methods for their preparation, pharmaceutical composition containing these compounds and their use in the treatment of complications of diabetes mellitus. The compounds of this series exhibit AGE breaking activity, which is essential for the treatment of diabetic and aging-related complications including kidney disease, nerve damage, atherosclerosis, retinopathy and dermatological conditions. The invention also extends to the method of reversing the discoloration of teeth resulting from nonenzymatic browning in the oral cavity which comprises administration of an amount effective to reverse pre-formed advanced glycosylation crosslinks.
2. Description of the Prior Art
Maillard in 1912 found that reducing sugars, such as glucose and ribose react with proteins to form brown pigments. Further studies have shown that this is an irreversible non-enzymatic reaction, which occurs in several natural systems including stored foodstuff. Maillard reaction occurs in two stages, early and advanced. Initially, proteins react with glucose to form stable Amadori products, which subsequently cross-links to form advanced glycation end products (AGE). In most cases, the formation of AGE also accompanies browning of the proteins and increase in the fluorescence.
In diabetes, where blood glucose level is significantly higher than normal, the reaction of glucose with several proteins such as haemoglobin, lens crystallin and collagen, gives rise to the formation of AGE, which in turn, is responsible for the complications associated with diabetes, such as nephropathy, microangiopathy, endothelial dysfunction and other organ dysfunctions. In addition, the activity of several growth factors, such as basic fibroblast growth factor, is also impaired. AGE products, unlike normal proteins in tissue, have a slower rate of turnover and replenishment. It has been reported that AGE products may in fact elicit a complex immunological reaction involving RAGE (Receptor for Advanced Glycation End Products) receptors and activation of several incompletely defined immunological processes. It has been documented that diabetes with evidence of microangiopathy and macroangiopathy also show evidence of oxidative stress, the mechanism of which has not been elucidated.
In vitro AGE formation can be studied in the laboratory by incubating reducing sugars, such as ribose or glucose with bovine serum albumin. AGE formation can be detected by increase in the fluorescence or increased cross reactivity with anti-AGE antibodies. The increase in fluorescence seems to precede formation of AGE specific antigenic epitopes. This increase in fluorescence is used to monitor the increased AGE formation in vitro (Brownlee M et al, Science 1986; 232:1629-1632). In addition to the increase in the fluorescence, one of the most important features of in vitro AGE formation is the formation of antigenic epitopes that are specific to AGE and not to the native proteins. Therefore, it is possible to raise antibodies against advanced glycation end products of one protein and use them to detect AGE formation in other proteins. This has served as an important analytical tool in AGE research.
Due to the clinical significance of AGE formation, many approaches are being used to diagnose, prevent, or revert AGE formation in the body. The formation of AGE could be inhibited by reacting with an early glycosylation product that results from the original reaction between the target protein and glucose. The inhibition was believed to take place as the reaction between the inhibitor and the early glycosylation product appeared to interrupt the subsequent reaction of the glycosylated protein with additional protein material to form the cross linked late stage product. Compounds like aminoguanidine act to inhibit AGE formation by such mechanism.
The formation of AGE on long-lived proteins is also associated with cross-linking of these proteins. The AGE derived protein cross-links have been shown to be cleaved by compounds like N-phenacyl thiazolium bromide (PTB), which reacts with and cleaves covalent, AGE derived protein cross links (Vasan et al. Nature 1996; 382: 275-278; U.S. Pat. No. 5,853,703, Date of Patent: Dec. 29, 1998). The mechanism of reducing the AGE content in tissues is expected to take place relatively rapidly, in contrast to aminoguanidine, which acts slowly by its very nature of mechanism of action. This current specification is related to compounds of pyridinium class, which break pre-formed AGE, like PTB, and in some cases even more effectively by than PTB.
The main objective of the present invention is to provide a new class of compounds of the pyridinium series which are useful for the management of diabetes and aging related vascular complications and particularly in the treatment of complications of diabetes mellitus and other aging related conditions including kidney disease, nerve damage, atherosclerosis, retinopathy and dermatological conditions. The invention also extends the method to reverse the discoloration of teeth resulting from nonenzymatic browning in the oral cavity which comprises administration of an amount effective to reverse the pre-formed advanced glycosylation crosslinks, etc.
Another object of the present invention is to provide compounds of the pyridinium series, which exhibit AGE breaking activities.
Yet another object of the present invention is to provide a method of preparation of compounds of the pyridinium series which exhibit AGE breaking activities.
Still another object of the invention is to provide pharmaceutical compositions with a new class of compounds of the pyridinium series according to the invention and their pharmaceutically acceptable salts in combination with suitable carriers, solvents, excepients, diluents and other media normally employed in preparing such compositions.
Still another object of the invention is to provide a method of treatment of a diabetic patient by administration of the compounds of the invention, either singly or in combination with drugs for anti-diabetic therapy, or pharmaceutically acceptable salts thereof in required dosage in admixture with pharmaceutically acceptable diluent, solvent, excepients, carriers or other media as may be appropriate for the purpose.
The present invention provides for a new class of AGE breakers, of general formula I, 
wherein
R1 is xe2x80x94R4-R5 or xe2x80x94N(R7)N (R7)R9;
R4 is selected from the group consisting of xe2x80x94N(R7)R6Oxe2x80x94, xe2x80x94N(R7)R6N(R7)xe2x80x94, OR6O, and xe2x80x94OR6N(R7)xe2x80x94,
where R6 is alkyl;
R5 is selected from the group consisting of alkyl, aryl including heteroaryl, xe2x80x94COR7, SO2R7, xe2x80x94C(S)NHR7, xe2x80x94C(NH)NHR7, xe2x80x94COR10, 
where R7 is selected from the group consisting of H, alkyl and aryl including heteroaryl provided R7 might be different for R1 and R3 in the same compound;
R2 is selected from the group consisting of F, Cl, Br, I, OR7, NO2, alkyl, aryl including heteroaryl, formyl, acyl, C(O)NR7R10, C(O)OR7, NR7R10, Nxe2x95x90C(R7)(R10), SR7, SO2NH2, SO2 alkyl and SO2aryl,
and m is 0, 1 or 2;
R3 is selected from the group consisting of R7, OR7, N(R7)(R10), Nxe2x95x90C(R7)(R10), N(R7)N(R7)(R10), N(R7) Nxe2x95x90C(R7)(R10) and CH(R7)C(O)R8 
where R8 is selected from the group consisting of R7, OR7 and NR7R10;
R9 is selected from the group consisting of hydrogen, alkyl, aryl including heteroaryl, C(O)R10, xe2x80x94SO2R10, xe2x80x94C(S)NHR10, xe2x80x94C(NH)NH(R10) and xe2x80x94C(O)NHR10,
R10 is selected for the group consisting of H, alkyl or aryl including heteroaryl and in each case optionally different from substituent R10, provided R10 might be different for R1 and R3 in the same compound;
X is selected from group consisting of a halide ion, acetate ion, perchlorate ion, sulfonate ion, oxalate ion, citrate ion, tosylate ion, maleate ion, mesylate ion, carbonate ion, sulfite ion, phosphoric hydrogen ion, phosphonate ion, phosphate ion, BF4xe2x88x92 and PF6xe2x88x92;
with proviso that,
(i) when two alkyl groups are present on the same carbon or nitrogen, they are optionally linked together to form a cyclic structure and
(ii) the nitrogen of heteroaryl ring of R10, when present, is optionally quaternized with compound such as Xxe2x80x94CH2C(O)xe2x80x94R3 
As used herein, xe2x80x9calkylxe2x80x9d refers to an optionally substituted hydrocarbon group joined by single carbon-carbon bonds and having 1 to 8 carbon atoms joined together. The alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. The substituents are selected from F, Cl, Br, I, N, S, O and aryl. Preferably, no more than three substituents are present.
As used herein xe2x80x9carylxe2x80x9d refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The substituents are selected from F, Cl, Br, I, N, S, O and straight chain or branched C1-C6 hydrocarbon.
The novel compounds of the invention of general formula I having m as 0 and xe2x80x94COR1 at position 3 are listed in Table 1A and the novel compounds of the invention of general formula I having m as 0 and xe2x80x94COR1 at position 4 are listed in Table 1B. The following compounds suggested are by way of example alone of the representative compounds of the general formula I as defined above and in no way restrict the invention.
N,Nxe2x80x2-Bis[3-carbonyl-1-(2-phenyl-2-oxoethyl)-pyridinium]hydrazine dibromide (compound 1):
N,Nxe2x80x2-Bis[3-carbonyl-1-(2-ethoxy-2-oxoethyl)pyridinium]hydrazine dibromide (compound 2):
N,Nxe2x80x2-Bis[3-carbonyl-1-(2-(2,4-dichlorophenyl)-2-oxoethyl)pyridinium]hydrazine dibromide (compound 3):
1-(2-Ethoxy-2-oxoethyl)-3-(2-(2-pyridyl)hydrazinocarbonyl)pyridinium bromide.hydrochloride (compound 4):
1-(2-Thien-2xe2x80x2-yl-2-oxoethyl)-3-(methanesulfonyl hydrazinocarbonyl)pyridinium bromide (compound 5):
N,Nxe2x80x2-Bis[3-carbonyl-1-(2-thien-2xe2x80x2-yl-2-oxoethyl)pyridinium]hydrazine dibromide (compound 6):
1-(2-Ethoxy-2-oxoethyl)-3-(2-(benzoyloxy)ethylaminocarbonyl)pyridinium bromide (compound 7):
1-(2-(2,4-Dichlorophenyl)-2-oxoethyl)-3-(2-(benzoyloxy)ethylamino-carbonyl)pyridinium bromide (compound 8):
1-(2-Thien-2xe2x80x2-yl-2-oxoethyl)-3-(2-(2-pyridyl)hydrazinocarbonyl)pyridinium bromide (compound 9):
1-(2-Phenyl-2-oxoethyl)-3-(2-(2-pyridyl)hydrazinocarbonyl)pyridinium bromide (compound 10):
1-(2-Phenyl-2-oxoethyl)-3-(hydrazinocarbonyl)pyridinium bromide (compound 11).
1-(2-Phenyl-2-oxoethyl)-3-(methanesulfonyl hydrazinocarbonyl)pyridinium bromide (compound 12):
1-(2-Ethoxy-2-oxoethyl)-3-(methanesulfonyl hydrazinocarbonyl)pyridinium bromide (compound 13):
1-(2-Phenyl-2-oxoethyl)-3-(phenylsulfonylhydrazino carbonyl)pyridinium bromide (compound 14):
1-(2-Phenyl-2-oxoethyl)-2-chloro-3-(phenylsulfonylhydrazino carbonyl)pyridinium bromide (compound 15):
1-(2-Phenyl-2-oxoethyl)-3-(2-(acetoxy)ethyloxy carbonyl)pyridinium bromide (compound 16):
1-(2-Ethoxy -2-oxoethyl)-3-(2-(benzoyloxy)ethyloxy carbonyl)pyridinium bromide (compound 17):
1-(2-Thien-2xe2x80x2-yl-2-oxoethyl)-4-(2-(benzoyloxy)ethylaminocarbonyl)pyridinium bromide (compound 18):
1-(2-Ethoxy -2-oxoethyl)-4-(phenylsulfonyl hydrazino carbonyl)pyridinium bromide (compound 19):
1-(2-Phenylamino-2-oxoethyl)-4-(phenylsulfonyl hydrazino carbonyl)pyridinium chloride (compound 20):
1-(2-Ethoxy -2-oxoethyl)-3-(phenylsulfonyl hydrazino carbonyl)pyridinium bromide (compound 21):
1-(2-(2,4-Dichlorophenyl)-2-oxoethyl)-3-(2(methoxy)ethyloxycarbonyl)pyridinium bromide (compound 22):
1-(2-Phenylamino-2-oxoethyl)-3-((benzoyloxy)ethylaminocarbonyl)pyridinium chloride (compound 23):
1-(2-Thien-2xe2x80x2-yl-2-oxoethyl)-3-(phenylaminocarbonyl hydrazinocarbonyl)pyridinium bromide (compound 24):
1-(2-Phenyl-2-oxoethyl)-3-(2-(acetoxy)ethylaminocarbonyl)pyridinium bromide (compound 25):
1-(2-Phenylamino-2-oxoethyl)-3-(phenyl sulfonyl hydrazino carbonyl)pyridinium chloride (compound 26):
1-(2-Phenylamino-2-oxoethyl)-3-((4-methylphenyl)sulfonyl hydrazino carbonyl)pyridinium chloride (compound 27):
1-(2-Phenyl-2-oxoethyl)-3-(2-(benzoyloxy)ethyloxy carbonyl)pyridinium bromide (compound 28):
1-(2-Thien-2xe2x80x2-yl-oxoethyl)-3-(phenylcarbonyl hydrazino carbonyl)pyridinium bromide (compound 29):
1-(2-Ethoxy-2-oxoethyl)-3-((phenylmethyl)sulfonyl hydrazino carbonyl)pyridinium bromide (compound 30):
1-(2-Phenyl-2-oxoethyl)-3-((phenylmethyl)sulfonyl hydrazino carbonyl)pyridinium bromide (compound 31):
According to the embodiment of the present invention, the present compounds are used for the treatment of diabetic complications, and aging related complications including kidney disease, nerve damage, atherosclerosis, retinopathy, dermatological conditions and colouration of teeth occurring due to the higher levels of preformed AGE. The increased levels of preformed AGE can be brought under control by breaking the AGE products using compounds mentioned in the invention.
The invention also provides a process for the preparation of novel compounds of the pyridinium series.
The said process for the preparation of compound 1, comprises, adding a solution of phenacyl bromide in isopropanol to N,Nxe2x80x2-bis-(nicotinyl)hydrazine dissolved in methanol, refluxing for six hours, cooling, filtering the precipitated solid, washing the solid with hot ethyl acetate and finally purifying the solid with 20 ml of methanol:ethyl acetate (3:1) to yield the desired compound.
Similarly, the other novel compounds of general formula I, are prepared from properly substituted pyridine derivatives followed by quarternization with appropriate reagent by refluxing in alcoholic solvents like, methanol, ethanol, propanol, etc and high boiling solvents like toluene or xylene etc, for 6-48 hrs. to give the desired compounds.
The in vitro AGE formation, studied in the laboratory, by incubating reducing sugar glucose, with protein bovine serum albumin, resulted in browning of solution and increase in the fluorescence. Fluorescence was used as the criteria to monitor the increased AGE formation.